The PAK inhibitor PF-3758309 sensitizes CD34⁺ nonresponder cells to TKIs and perturbs mitochondrial activity. (A) Representative confocal images showing the morphology of mitochondria stained in CD34⁺ IM nonresponder cells treated with PF-3758309 (PF) and IM, as compared with control and single drug treatment and quantification of mitochondria to nuclear area ratio in these treated cells are shown and each dot represents an individual cell (top, n = 2). The white scale bar represents 20 μm. Intracellular mitochondrial staining of the same cells by MitoTracker Deep Red (MTDR) is also shown (bottom). (B) ROS was assessed using CellROX Deep Red in the same cells as in (A), and representative images and intracellular ROS accumulation are shown. The white scale bar represents 50 μm. (C-D) Viability and apoptosis assays of IM-resistant K562 cells (K562R) and CD34⁺ cells from IM nonresponders (n = 3) for 72 hours in the presence or absence of PF or TKIs (IM, DA, and NL), alone or in combination. (E) CFC assays of CD34⁺ CML cells from TKI nonresponders (n = 3) with or without PF or TKIs, alone or in combination. Data shown are mean ± SEM of measurements from 3 patient samples. P values were calculated using a 2-tailed unpaired Student t test. (F) Model of how restored expression of miR-185 or inhibition of PAK6 activity sensitizes TKI-resistant CML cells to TKIs both in vitro and in vivo, by dual targeting of a novel miR-185-PAK6 axis and BCR-ABL1 activity.